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          植物总RNA提取
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              <time title="创建时间：2020-10-09 00:15:06 / 修改时间：00:38:56" itemprop="dateCreated datePublished" datetime="2020-10-09T00:15:06+08:00">2020-10-09</time>
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<h2 id="实验材料、试剂及器材"><a href="#实验材料、试剂及器材" class="headerlink" title="实验材料、试剂及器材"></a>实验材料、试剂及器材</h2><h3 id="材料"><a href="#材料" class="headerlink" title="材料"></a>材料</h3><p>二穗短柄草或小麦幼苗</p>
<h3 id="试剂"><a href="#试剂" class="headerlink" title="试剂"></a>试剂</h3><ul>
<li>总RNA提取纯化试剂Trizol、75%乙醇、异丙醇、氯仿、液氮，</li>
<li>RNA酶抑制蛋白(Recombinant RNase Inhibitor) ：40 U/uL</li>
<li>dNTP 混合物：10 mM、2.5 mM</li>
<li>Oligo(dT) 18：250ng /uL</li>
<li>5x逆转录合成缓冲液</li>
<li>MMLV逆转录酶（PrimeScript Reverse Transcriptase）： 200U/uL</li>
<li>actin引物：上游和下游各10uM</li>
<li>Tap DNA聚合酶：5U/uL</li>
<li>10 x PCR缓冲液</li>
<li>DEPC水</li>
</ul>
<h3 id="器材"><a href="#器材" class="headerlink" title="器材"></a>器材</h3><p>研钵、1.5ml离心管（RNase-Free）、枪头、移液枪、Nanodrop超微量分光光度计、PCR扩增仪、高速冷冻离心机、水平电泳仪、凝胶成像系统、恒温金属浴。</p>
<h2 id="实验前准备"><a href="#实验前准备" class="headerlink" title="实验前准备"></a>实验前准备</h2><ol>
<li>所有的玻璃器皿和陶瓷制品应在使用前，用锡纸包住，180 ℃干烘3小时，烘烤完毕后将烘箱调回以前设置温度并关闭电源，待冷却后取出，整个过程中，在使用前都应保持锡纸是封紧的。</li>
<li>离心管和枪头用0.1%预处理过的DEPC水浸泡过夜，沥干后装入RNA提取专用灭菌塑料袋和枪头盒，然后高压灭菌20分钟，以去除残留的DEPC（进口离心管和枪头可不用DEPC水处理，灭菌后即可用）。</li>
<li>配制的溶液如不能高压灭菌, 应尽可能使用0.1%预处理过的DEPC水配制，并尽可能使用未曾开封的试剂。</li>
</ol>
<h2 id="操作步骤"><a href="#操作步骤" class="headerlink" title="操作步骤"></a>操作步骤</h2><h3 id="植物总RNA的提取"><a href="#植物总RNA的提取" class="headerlink" title="植物总RNA的提取"></a>植物总RNA的提取</h3><ol>
<li>取50-100 mg样品在液氮中研磨成粉末(越细越好)，并尽可能多地转入1.5 ml离心管中;</li>
<li>然后加入1ml Trizol植物组织裂解液，剧烈震荡混匀，室温静置1min（注：样品总体积不能超过所用Trizol体积的10%）（以下其它抽提试剂的加入量为对应加入1 ml Trizol试剂的标准量，具体加入量可按此比例酌情增减）；</li>
<li>4 ℃，12000 g离心10min，取上清（900μl）转入新的离心管中，室温静置5min；</li>
<li>在通风柜内加入300 μl氯仿，剧烈摇匀1min，使之快速溶解于裂解液中，室温静置3min；</li>
<li>4 ℃，12000 g离心15min，取上清无色水相（上层）转入一新的1.5ml离心管中（400μl），加入等体积氯仿，剧烈摇匀1min，室温静置3min（注：底层为氯仿相，中层为蛋白相，上层是无色水相，含RNA）；</li>
<li>4 ℃，12000 g离心10min，将无色水相转入一新的离心管中（不要贪多，300μl即可，吸取上清的时候一定要特别小心，不要把中间蛋白层吸上来），加入等体积异丙醇于上清，用力摇匀，室温静置15min，4 ℃，12000 g离心10min；</li>
<li>小心弃上清，加入1 mL 75%乙醇（DEPC水配制），用枪头将沉淀捣烂（目的是更彻底地清洗RNA，溶解杂质，不要把沉淀吸入枪头中扔掉），4 ℃，12000g离心5min，小心弃上清；</li>
<li>重复步骤7一次</li>
<li>短暂离心，小心吸去剩余上清；</li>
<li>将离心管置超净台上空气自然干燥10-30min（时间自己把握，RNA沉淀透明即可）；</li>
<li>用20μl DEPC水溶解，用Nanodrop核酸测量仪测定浓度。</li>
</ol>
<h3 id="庄盟生物植物组织RNA提取试剂盒（ZP405）操作步骤："><a href="#庄盟生物植物组织RNA提取试剂盒（ZP405）操作步骤：" class="headerlink" title="庄盟生物植物组织RNA提取试剂盒（ZP405）操作步骤："></a>庄盟生物植物组织RNA提取试剂盒（ZP405）操作步骤：</h3><p>提示：第一次使用前请先在漂洗液RW中加入指定量乙醇！</p>
<ol>
<li><p>匀浆处理每20-80mg幼嫩植物组织加1ml植物裂解液R，用研磨杵将组织彻底研磨（如组织较难彻底研磨，可选用电动或玻璃匀浆器）；或者取30-80mg幼嫩植物组织加液氮彻底研磨后，转入到已经加有1m植物裂解液R的1.5m离心管中，涡旋振荡混匀。</p>
</li>
<li><p>可选步骤：4℃的条件下12000rpm离心10分钟，小心取上清转入一个新的 RNase-free的离心管中。</p>
<blockquote>
<p>当样品富含蛋白质、脂肪、多糖或是细胞外物质，例如植物的块茎部分时可能需要这一分离步骤。匀浆化后在2-8°C的条件下以12000rpm离心10分钟，移除匀浆中不溶解的物质，余下的沉淀中包含有细胞外膜、多糖以及高分子量DNA，而上层的超浮游物含有RNA。</p>
</blockquote>
</li>
<li><p>每1ml植物裂解液R加0.2ml氯仿。盖紧样品管盖，剧烈振荡（涡旋亦可）10秒并将其在室温下孵育5分钟。</p>
</li>
<li><p>于4°C12000rpm离心10分钟，样品会分成三层：下层有机相、中间层和上层无色的水相，RNA存在于水相中。水相层的容量大约为所加裂解液R体积的60%，把水相转移到新管中，进行下一步操作。</p>
<blockquote>
<p>建议：</p>
<ul>
<li>分层后细胞裂解液对RNA的保护作用减弱，因此操作上低温快速更好。</li>
<li>由正中插入液面下缓慢吸取大约500μl上相，避免扰动带 RNase的中间层一般吸取大约400-500μl上相即可，不必要尽可能多的吸取含RNA的水相。</li>
</ul>
</blockquote>
</li>
<li><p>加入0.5倍体积的无水乙醇和100μl结合液N，颠倒混匀（此时可能会出现沉淀）。得到的溶液和可能沉淀一起转入吸附柱中，吸附柱套在收集管内。</p>
</li>
<li><p>12000rpm离心30秒，弃掉废液，将吸附柱重新套回收集管。</p>
</li>
<li><p>加入500μl漂洗液RW（请先检查是否已加入无水乙醇！），12000rpm离心30秒，弃掉废液。</p>
</li>
<li><p>重复步骤7。</p>
</li>
<li><p>将吸附柱放回空收集管中，13000rpm离心2分钟，尽量除去漂洗液，以免漂洗液中残留乙醇抑制下游反应。</p>
</li>
<li><p>取出吸附柱，放入一个新的 RNase-free离心管中，根据预期RNA产量在吸附膜的中间部位加60-100μl RNase-free water，室温放置2分钟，12000pm离心1分钟，得到RNA溶液。</p>
<blockquote>
<p>洗脱体积越大，洗脱效率越高，如果需要RNA浓度较高，可以适当减少洗脱体积，但是最小体积最好不少于50μl，体积过小降低RNA洗脱效率，减少RNA产量</p>
</blockquote>
</li>
<li><p>电泳推荐方案：用3mm×1mm小梳子，初始量为5μl电泳。电泳电压4-10v/cm，电泳时间20-25分钟。</p>
</li>
</ol>
<h2 id="注意事项"><a href="#注意事项" class="headerlink" title="注意事项"></a>注意事项</h2><ol>
<li>第一次使用前请先在漂洗液RW瓶中加入指定量乙醇，加入后请及时打钩标记已加入乙醇，以免多次加入！</li>
<li>为防止RNA降解，所有离心步骤如未加说明，均在4°C低温进行。使用转速可以达到13000pm的冷冻离心机即可。</li>
<li>裂解液R中含有刺激性有害化合物，操作时要戴乳胶手套，避免沾染皮肤、眼晴和衣服。若沾染皮肤、眼睛时，要用大量清水或者生理盐水冲洗。</li>
<li>本试剂盒不含有实验室常用试剂氯仿，用户使用前需要自备氯伤。</li>
<li>常规的琼脂糖凝胶电泳和变性胶电泳均可以用来分析RNA的质量。好的RNA产物在电泳后应该可以看到明显的二条优势核糖体RNA带，分别为~5kb（28S），~2kb（18S），条带亮度比值约为2:1.有时候也可以看到~0.kb和0.3kb（5s，tRNA）带。但有时候根据不同的物种如某些植物组织可以看到4、5条带也属于正常现象，如果RNA未成熟的前体或者不均一核RNA、小核RNA提取出来也可能看到介于7kb和15kb之间的不连续的高分子量条带。</li>
<li>检测OD<sub>260</sub>/OD<sub>280</sub>吸光度比值时，RNA样品应该溶于TE后检测，如果用水稀释后检测由于一般水离子强度和PH值低，会使OD<sub>280</sub>升高，从而使比值降低。</li>
<li>加入裂解液R匀浆后，加氯仿前，样品可在-60°C~-70℃保存一个月以上。</li>
</ol>
<p>附：RNA样品指标：</p>
<p>OD<sub>260</sub>/OD<sub>280</sub>≥1.8 去除蛋白指标；</p>
<p>如果OD<sub>260</sub>/OD<sub>280</sub>比值均在1.8-2.0之间，说明RNA的纯度很好。</p>
<p>1.8以下说明有蛋白或DNA污染，2.0以上说明RNA有不同程度的降解。</p>
<p>OD<sub>260</sub>/OD<sub>230</sub>≥2.0 去除盐分指标。</p>

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